Bone pastes comprising biofunctionalized calcium phosphate cements with enhanced cell functions for bone repair

ABSTRACT

The invention provides injectable, biofunctional agent-containing calcium phosphate cement bone pastes for bone tissue engineering, and methods of making and using the same.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. DE014190awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

The invention provides injectable, biofunctional agent-containingcalcium phosphate cement bone pastes for bone tissue engineering, andmethods of making and using the same.

BACKGROUND

Bone transplantation is used to treat bone defects arising from trauma,disease, congenital deformity, or tumor resection. Bone is the secondmost transplanted tissue after blood; however, current bonetransplantation methods involve certain disadvantages. For example, boneautografts (i.e., bone harvested from a patient to be treated) are oflimited availability and incur donor site morbidity. Bone allografts(i.e., bone harvested from a person who is not the patient to betreated) carry a risk of disease transmission. With seven million bonefractures per year in the U.S., and musculoskeletal conditions costing$215 billion [1,2], new biomaterial treatments are needed.

Calcium phosphate cements (CPCs) can be molded and set in situ to formhydroxyapatite, they are highly osteoconductive, and they can beresorbed and replaced by new bone. Recent studies have developed CPCinto a carrier for stem cell delivery to enhance bone regeneration. Stemcell-based tissue engineering has immense potential to regeneratedamaged and diseased tissues [3-7].

Human bone marrow mesenchymal stem cells (hBMSCs) can differentiate intoosteoblasts, adipocytes, chondrocytes, myoblasts, neurons, andfibroblasts [8-10]. hBMSCs can be harvested from a patient, expanded inculture, induced to differentiate, and combined with a scaffold torepair bone defects. However, harvesting of autogenous hBMSCs requiresan invasive procedure. Moreover, autogenous hBMSCs display lowerself-renewal potential with aging of the individual from whom the cellsare obtained.

Human umbilical cord mesenchymal stem cells (hUCMSCs) have been used intissue engineering [11-16]. Umbilical cords can provide an inexpensiveand inexhaustible stem cell source, without the invasive procedure ofhBMSCs, and without the controversies of embryonic stem cells (hESCs).hUCMSCs are primitive MSCs that exhibit a high plasticity anddevelopmental flexibility and appear to cause no immunorejection in vivo[12]. hUCMSCs have been cultured on tissue culture plastic [13], polymerscaffolds [16], and calcium phosphate scaffolds for tissue engineering[17-19].

Calcium phosphate (CaP) scaffolds are important for bone repair becausethey are bioactive, mimic the bone minerals, and can bond to neighboringbone, in contrast to bioinert implants that can form undesirable fibrouscapsules [20-22]. The CaP minerals provide a preferred substrate forcell attachment and expression of the osteoblast phenotype [23,24].However, for pre-formed bioceramic scaffolds to fit into a bone cavity,a surgeon must machine the graft or carve the surgical site, leading toincreases in bone loss, trauma, and surgical time [2]. Pre-formedscaffolds have other drawbacks, including the difficulty in seedingcells deeply into the scaffold and the inability to inject suchscaffolds in minimally-invasive surgeries [2,10].

Injectable scaffolds for cell delivery are advantageous because theycan: (i) shorten the surgical operation time; (ii) minimize the damagingeffects of large muscle retraction; (iii) reduce postoperative pain andscar size; (iv) achieve rapid recovery; and (v) reduce cost. Variousinjectable hydrogel and polymer carriers can be used for stem celldelivery [10,25]. However, current injectable carriers cannot be used inload-bearing repairs [10,25], such as those required in bone. Forexample, hydrogel scaffolds do not possess the mechanical strength to beused in load bearing applications [25].

Mechanical properties of scaffolding materials are of crucial importancein regeneration of load-bearing tissues such as bone. Specifically,scaffolding materials must be able to withstand stresses to avoidscaffold fracture and to maintain scaffold structure to define the shapeof the regenerated tissue. However, to date, an injectable, bioactive,and strong scaffold for stem cell encapsulation and bone engineering hasnot yet been developed.

Hydroxyapatite (HA) and other calcium phosphate (CaP) bioceramics areuseful in hard tissue repair because of their excellent biocompatibility[5,8,10,20-24]. When implanted into an osseous site, bone bioactivematerials such as HA and other CaP implants and coatings provide anideal environment for cellular reaction and colonization by osteoblasts.This leads to a tissue response termed osteoconduction in which bonegrows on and bonds to the implant, promoting a functional interface.These bioceramics are highly useful for bone repair. However, onedrawback is that sintered HA implants are generally not resorbable.Another limitation is that these bioceramics are pre-forms that requiremachining and may leave gaps when fitted into a bone cavity.

In contrast to CaP bioceramics, calcium phosphate cements (CPCs) canself-set in the bone site with intimate adaptation to complex shapes,they can be easily contoured for esthetics in craniofacial repairs, andthey are highly osteoconductive and bioresorbable [26-32]. CPCs can beinjected or molded, and set in situ to form a bioactive scaffold thatbonds to bone [26-29]. The first CPC was approved by the Food and DrugAdministration (FDA) in 1996 for craniofacial repairs [26,30-32]. CPChas excellent osteoconductivity and can be replaced by new bone [30-32].However, several previous studies showed that human stem cell attachmenton CPC is relatively poor. Therefore, there is a need to improve thecell attachment to CPC to enhance bone repair efficacy.

SUMMARY

Provided herein are injectable, self-setting, biofunctionalagent-containing, and mechanically-strong bone pastes for bone tissueengineering. The bone pastes employ a calcium phosphate cement and oneor more biofunctional agents, and optionally, additional components,such as chitosan and/or fibers for reinforcement, and a porogen toincrease porosity of the pastes. In a further optional aspect, the bonepastes include cells or cell-encapsulating microbeads. In particularfurther aspects, the cells are stem cells.

In particular, and in a first aspect, described herein are bone pastescomprising a calcium phosphate cement and one or more biofunctionalagents. The biofunctional agents include RGD-containing peptides,fibronectin, fibronectin-like engineered polymer protein (FEPP),extracellular matrix (ECM), and platelet concentrate. Thus, in a firstembodiment, the bone paste comprises calcium phosphate cement and aRGD-containing peptide. The RGD-containing peptide is present within arange of about 0.0005% to about 5% by mass. The RGD-containing peptidecan be, e.g., RGD, G4RGDSP, RGDS, GRGD, GRGDGY, RGDSGGC, GRGDS, oranother RGD-containing polypeptide. In a second embodiment, the bonepaste comprises calcium phosphate cement and fibronectin. Thefibronectin is present within a range of about 0.0005% to about 5% bymass. In a third embodiment, the bone paste comprises calcium phosphatecement and FEPP. The FEPP is present within a range of about 0.0005% toabout 5% by mass. In a fourth embodiment, the bone paste comprisescalcium phosphate cement and ECM. The ECM is present within a range ofabout 0.001% to about 10% by mass. The ECM may be derived ECM (dECM). Ina fifth embodiment, the bone paste comprises calcium phosphate cementand platelet concentrate. The platelet concentrate is present within arange of about 0.001% to about 10% by mass. In further embodiments, thebone paste comprises calcium phosphate cement and any two of thebiofunctional agents, or any three of the biofunctional agents, or anyfour of the biofunctional agents, or each of the five biofunctionalagents.

The calcium phosphate cement of the bone paste may comprise, forexample, but not be limited to, one or more ingredients selected fromthe group consisting of tetracalcium phosphate (TTCP) (Ca₄(PO₄)₂O),dicalcium phosphate anhydrous (DCPA) (CaHPO₄), dicalcium phosphatedihydrate (CaHPO₄.2H₂O), tricalcium phosphate (Ca₃[PO₄]₂), α-tricalciumphosphate (α-Ca₃(PO₄)₂), β-tricalcium phosphate (β-Ca₃(PO₄)₂),octacalcium phosphate (Ca₈H₂(PO₄)₆.5H₂O), amorphous calcium phosphate(Ca₃(PO₄)₂), calcium carbonate (CaCO₃), calcium hydroxide (Ca[OH]₂), andhydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), and mixtures thereof.

In one embodiment, the calcium phosphate cement comprises tetracalciumphosphate and dicalcium phosphate anhydrous. In this embodiment, thecalcium phosphate cement may comprise, for example, but not be limitedto, a molar ratio of tetracalcium phosphate to dicalcium phosphateanhydrous of about 1:5 to about 5:1, a molar ratio of tetracalciumphosphate to dicalcium phosphate anhydrous of about 1:3 to about 1:1, orthe calcium phosphate cement comprises an approximately 1:1 molar ratioof tetracalcium phosphate to dicalcium phosphate anhydrous.

The calcium phosphate cement of the bone paste may further comprisechitosan. When chitosan is present, the RGD-containing peptide may becovalently linked to the chitosan.

The calcium phosphate cement of the bone paste may further comprisefibers. The fibers may be, but are not limited to, degradable fibers.

The calcium phosphate cement of the bone paste may further comprisechitosan and fibers, including, but not limited to, degradable fibers.

The calcium phosphate cement of the bone paste may further comprise aporogen. As an example, but not a limitation, the porogen may be NaHCO₃and citric acid. When the porogen is NaHCO₃ and citric acid, the massfraction of NaHCO₃ may be from about 5% to about 30%, and the massfraction of citric acid may be from about 50% to about 60%.

The bone paste may further comprise a bioactive agent. The bone pastemay also be injectable.

The bone paste may further comprise cells. When present, the cellsinclude, but are not limited to, human umbilical cord mesenchymal stemcells, bone marrow stem cells, embryonic stem cells, pluripotent stemcells, induced pluripotent stem cells, multipotent stem cells,progenitor cells, and osteoblasts. The cells may be attached to asurface of the bone paste, or the cells may be interspersed throughoutthe bone paste, or both.

In a second aspect, described herein are bone pastes comprising acalcium phosphate cement, one or more biofunctional agents, andcell-encapsulating microbeads. The biofunctional agents includeRGD-containing peptides, fibronectin, fibronectin-like engineeredpolymer protein (FEPP), extracellular matrix (dECM), and plateletconcentrate. Thus, in a first embodiment, the bone paste comprisescalcium phosphate cement, a RGD-containing peptide, andcell-encapsulating microbeads. The RGD-containing peptide is presentwithin a range of about 0.0005% to about 5% by mass. The RGD-containingpeptide can be, e.g., RGD, G4RGDSP, RGDS, GRGD, GRGDGY, RGDSGGC, GRGDS,or another RGD-containing peptide. In a second embodiment, the bonepaste comprises calcium phosphate cement, fibronectin, andcell-encapsulating microbeads. The fibronectin is present within a rangeof about 0.0005% to about 5% by mass. In a third embodiment, the bonepaste comprises calcium phosphate cement, FEPP, and cell-encapsulatingmicrobeads. The FEPP is present within a range of about 0.0005% to about5% by mass. In a fourth embodiment, the bone paste comprises calciumphosphate cement, ECM, and cell-encapsulating microbeads. The ECM ispresent within a range of about 0.001% to about 10% by mass. The ECM maybe derived ECM (dECM). In a fifth embodiment, the bone paste comprisescalcium phosphate cement, platelet concentrate, and cell-encapsulatingmicrobeads. The platelet concentrate is present within a range of about0.001% to about 10% by mass. In further embodiments, the bone pastecomprises calcium phosphate cement, cell-encapsulating microbeads, andany two of the biofunctional agents, or any three of the biofunctionalagents, or any four of the biofunctional agents, or each of the fivebiofunctional agents.

The calcium phosphate cement of the bone paste may comprise, forexample, but not be limited to, one or more ingredients selected fromthe group consisting of tetracalcium phosphate (TTCP) (Ca₄(PO₄)₂O),dicalcium phosphate anhydrous (DCPA) (CaHPO₄), dicalcium phosphatedihydrate (CaHPO₄.2H₂O), tricalcium phosphate (Ca₃[PO₄]₂), α-tricalciumphosphate (α-Ca₃(PO₄)₂), β-tricalcium phosphate (β-Ca₃(PO₄)₂),octacalcium phosphate (Ca₈H₂(PO₄)₆.5H₂O), amorphous calcium phosphate(Ca₃(PO₄)₂), calcium carbonate (CaCO₃), calcium hydroxide (Ca[OH]₂), andhydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), and mixtures thereof.

In one embodiment, the calcium phosphate cement comprises tetracalciumphosphate and dicalcium phosphate anhydrous. In this embodiment, thecalcium phosphate cement may comprise, for example, but not be limitedto, a molar ratio of tetracalcium phosphate to dicalcium phosphateanhydrous of about 1:5 to about 5:1, a molar ratio of tetracalciumphosphate to dicalcium phosphate anhydrous of about 1:3 to about 1:1, orthe calcium phosphate cement comprises an approximately 1:1 molar ratioof tetracalcium phosphate to dicalcium phosphate anhydrous.

The calcium phosphate cement of the bone paste may further comprisechitosan. When chitosan is present, the RGD-containing peptide may becovalently linked to the chitosan.

The calcium phosphate cement of the bone paste may further comprisefibers. The fibers may be, but are not limited to, degradable fibers.

The calcium phosphate cement of the bone paste may further comprisechitosan and fibers, including, but not limited to, degradable fibers.

The calcium phosphate cement of the bone paste may further comprise aporogen. As an example, but not a limitation, the porogen may be NaHCO₃and citric acid. When the porogen is NaHCO₃ and citric acid, the massfraction of NaHCO₃ may be from about 5% to about 30%, and the massfraction of citric acid may be from about 50% to about 60%.

The bone paste may further comprise a bioactive agent. The bone pastemay also be injectable.

The microbeads of the bone paste can include hydrogel microbeads, forexample, but not limited to, microbeads comprising alginate, partiallyoxidized alginate, oxidized alginate, alginate-fibrin, partiallyoxidized alginate-fibrin, oxidized alginate-fibrin, poly(ethylene glycoldiacrylate), poly(ethylene glycol)-anhydride dimethacrylate, gelatin,chemically cross-linked polymers, ionically cross-linked polymers,heat-polymerized polymers, or photopolymerized polymers. In oneembodiment, the microbeads are alginate-fibrin microbeads. When themicrobeads are alginate-fibrin microbeads they may comprise a fibrinogenmass fraction of from about 0.05% to about 1%, such as, but not limitedto, a fibrinogen mass fraction of about 0.1%. The alginate may be atabout 7.5% oxidation.

The microbeads of the bone paste may be present in a volume of about 40to 60%, and they may have an average diameter of less than about 2millimeters.

The cells of the bone paste may be stem cells, for example, but notlimited to, one or more of human umbilical cord mesenchymal stem cells,bone marrow stem cells, stem cells from breast milk or other bodyfluids, embryonic stem cells, pluripotent stem cells, inducedpluripotent stem cells, multipotent stem cells, progenitor cells, andosteoblasts.

The bone paste may further comprise a bioactive agent. The bone pastemay also be injectable.

In a third aspect, described herein is a method for preparing a bonepaste, comprising covalently linking an RGD-containing peptide tochitosan to form RGD-grafted chitosan, dissolving the RGD-graftedchitosan in water to form a chitosan liquid, and mixing calciumphosphate cement into the chitosan liquid, thereby preparing a bonepaste. The RGD-containing peptide can be, e.g., RGD, G4RGDSP, RGDS,GRGD, GRGDGY, RGDSGGC, GRGDS, or another RGD-containing peptide. TheRGD-containing peptide is present within a range of about 0.0005% toabout 5% by mass. The chitosan may be chitosan lactate. A degradablefiber may be added to the chitosan liquid prior to mixing calciumphosphate cement into the chitosan liquid. The calcium phosphate cementmay comprise an approximately 1:1 molar ratio of tetracalcium phosphateto dicalcium phosphate anhydrous.

In a fourth aspect, described herein is a method of repairing orremodeling a bone, comprising administering to a bone an effectiveamount of any of the bone pastes described herein and allowing the bonepaste to harden, thereby repairing or remodeling the bone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the physical properties of biofunctionalized CPCs. (A)Cement setting time (mean±sd; n=3), (B) flexural strength (mean±sd;n=6), and (C) elastic modulus (mean±sd; n=6). In each plot, bars ofvalues with dissimilar letters are significantly different (p<0.05).

FIG. 2 shows live/dead staining photos of hUCMSCs on biofunctionalizedCPC. Live cells showed green fluorescence. Dead cells were stained red.There were numerous live cells and very few dead cells (not shown). At 1d, there was noticeably more cell attachment to biofunctionalized CPCsthan CPC control. Cell density was significantly increased from 1 d to 8d due to proliferation. There were noticeably more cells onbiofunctionalized CPCs than on CPC control.

FIG. 3 shows hUCMSC viability on biofunctionalized CPCs: (A) Live celldensity, and (B) percentage of live cells. Each value is mean±sd; n=5.In each plot, values with dissimilar letters are significantly different(p<0.05).

FIG. 4 shows fluorescence of actin fibers in hUCMSCs onbiofunctionalized CPCs. (A-F) CPC-RGD, CPC-Fn, CPC-FEPP, CPC-GELTREX™,and CPC-Platelets, respectively. The actin stress fibers in the hUCMSCswere stained red. The cell nuclei (blue fluorescence) indicated thelocation and distribution of the hUCMSCs on the scaffold. The red colorwas brighter and denser with the addition of biofunctional agents inCPC. (G) Actin fiber fluorescence area fraction. The area offluorescence for actin fibers was divided by the total area of thephoto. Each value is mean±sd; n=5. Values with dissimilar letters aresignificantly different (p<0.05).

FIG. 5 shows RT-PCR of osteogenic differentiation of hUCMSCs onbiofunctionalized CPCs. (A) ALP, (B) Runx2, (C) OC, (D) collagen I geneexpressions. Each value is mean±sd; n=5. In each plot, values withdissimilar letters are significantly different (p<0.05).

FIG. 6 shows mineral synthesis by hUCMSCs on biofunctionalized CPCs. ARSstained minerals into a red color. (A) ARS staining of hUCMSC-scaffoldconstructs after 4 d and 21 d, on CPC control, CPC-Fn, andCPC-Platelets, as examples. For all materials, the mineral stainingbecame a thicker and darker red with increasing time from 4 d to 21 d.Between materials, mineralization was thicker and denser onbiofunctionalized CPCs than that on CPC control. (B) Mineralconcentration synthesized by the hUCMSCs was measured by an osteogenesisassay (mean±sd; n=3). Dissimilar letters at the bars indicate valuesthat are significantly different (p<0.05).

FIG. 7 shows hUCMSC-encapsulating alginate-fibrin microbeads embeddedpartially into biofunctionalized CPC surface. (A-D) Schematicillustrating microbeads in CPC with cell release at 1 d, 7 d, 14 d and21 d, respectively. (E, F) Optical photos of microbeads at 1 d and 14 d,respectively. A blue filter was used to enhance the contrast and clarityof microbeads. At 1 d, there was no microbead degradation, and theencapsulated cells appeared as rounded dots. At 14 d, the microbeadsdegraded and the hUCMSCs were released, showing cell attachment withelongated and spreading morphology.

FIG. 8 shows live/dead staining photos of hUCMSC-encapsulatingmicrobeads in the CPC surface. The scaffold type is listed on the top ofeach column: CPC control, CPC-mixed-Fn, CPC-mixed-RGD, andCPC-grafted-RGD. The culture times are listed on the left side for eachrow: 1 d, 7 d, 14 d and 21 d. The first four rows show live cells(stained green). The last row shows dead cells (red). There werenumerous live cells, and few dead cells. Incorporation of Fn and RGDinto CPC increased cell attachment and proliferation. CPC control hadthe least cells. CPC-grafted-RGD appeared to have the most cells.

FIG. 9 shows quantitative cell viability of hUCMSCs released from themicrobeads and attached to CPC. (A) Percentage of live cells, and (B)live cell density. CPC-grafted-RGD had the highest percentages of livecells. CPC-grafted-RGD had the most live cell density per mm2. Eachvalue is the mean of five measurements, with the error bar showing onestandard deviation (mean±sd; n=5).

FIG. 10 shows osteogenic differentiation of hUCMSC-encapsulatingmicrobeads in CPC-grafted-RGD surface. (A) ALP, (B) OC, (C) collagentype I, and (D) Runx2 gene expressions, measured by RT-PCR. All fourmarkers reached much higher levels than 1 d, indicating successfulosteogenic differentiation of the hUCMSCs released from the microbeadsand attached to the CPC-grafted-RGD scaffold. Each value is mean±sd;n=5.

FIG. 11 shows bone mineral synthesis by hUCMSCs released from themicrobeads and attached to the CPC-grafted-RGD disks. (A) Disks withoutcells were immersed as control. (B) Disk with cells at 7 d. (C) Diskwith cells at 14 d. (D) Disk with cells at 21 d. The red staining becamemuch thicker and denser over time, and the layer of new mineral matrixsynthesized by the cells covered the entire disk at 21 d. (E)Cell-synthesized mineral concentration was measured by the osteogenesisassay. Each value is mean±sd; n=5.

FIG. 12 shows mechanical properties of biofunctionalized CPC containingvarious biofunctional molecules. (A) Flexural strength, (B) elasticmodulus, and (C) work-of-fracture (toughness). Each value is mean±sd;n=5.

DETAILED DESCRIPTION

The need for bone regeneration or remodeling can arise from trauma,disease, congenital deformity, or tumor resection. Stem cell-scaffoldapproaches hold immense promise for bone tissue engineering. Currently,pre-formed scaffolds for cell delivery have drawbacks, including thedifficulty of seeding cells deeply into the scaffold and the inabilityto inject such scaffolds in repair procedures involving minimallyinvasive surgeries. Current injectable polymeric carriers and hydrogelsare too weak for load-bearing orthopedic application.

Provided herein is an injectable, strong, biofunctional agent-containingbone paste for bone tissue engineering. In the bone pastes describedherein, a calcium phosphate cement (CPC) is combined with one or morebiofunctional agents. The bone pastes may optionally further comprisecells or cell-encapsulating (e.g., hydrogel) microbeads, where the cellsinclude human umbilical cord mesenchymal stem cells (hUCMSCs) or stemcells from other sources. The CPC may optionally contain chitosan and/ordegradable fibers for reinforcement. The CPC may further optionallycontain a porogen to increase the porosity of the bone paste. Theresulting bone pastes are fully injectable under small injection forces.When cell-encapsulating microbeads are present in the bone paste, thecell viability post-injection matches that in hydrogel without CPC andwithout injection. Stem cells in the injectable bone pastes maintaintheir ability to osteodifferentiate, as indicated by expression ofosteogenic markers such as alkaline phosphatase and osteocalcin and byaccumulation of bone minerals. Mechanical properties of the bone pastesmatch the reported values of cancellous bone, and are much higher thanprevious injectable polymeric and hydrogel carriers.

The addition of biofunctional agents such as fibronectin and Arg-Gly-Asp(RGD) peptide improves cell attachment and proliferation [36-45]. Anycombination of one or more of the five classes of biofunctional agentsmay be incorporated into the bone pastes of the present invention. Thefirst class is the RGD-containing peptides, i.e., peptides that containthe sequence RGD, which is a known integrin-recognition site thatpromotes cell attachment. The second class is fibronectin (Fn), which isa general cell adhesion molecule that anchors cells to collagen andproteoglycan. The third class is fibronectin-like engineered proteinpolymer (FEPP). The fourth class is extracellular matrix (ECM). Thefifth class is platelet concentrate, a fraction of the plasma in whichplatelets are concentrated.

Bone pastes comprising one of these five classes of biofunctional agentsare generally referenced herein as: CPC-RGD, CPC-Fn, CPC-FEPP, CPC-ECM,and CPC-Platelets. Each of these five different types of bone pastesubstantially enhances stem cell functions, such as those of hUCMSCs.Live cell density and actin stress fibers are greatly increased due tothe incorporation of biofunctional agents in CPC. Actin stress fibersanchor to the cell membrane at locations that are frequently connectedto the ECM or the scaffold, and these connection sites are called focaladhesions [58]. The mineralization by hUCMSCs is also markedly enhancedby incorporation of biofunctional agents in CPC. Therefore, thebiofunctionalized CPCs used in the bone pastes of the present inventionshould greatly enhance cell function and bone regeneration.

The hUCMSC-microbead-CPC bone pastes described herein are fullyinjectable; when included, chitosan and fibers increase the mechanicalproperties, without compromising the injectability, of the pastes. Theinjection process does not interfere with the osteogenic capacity of thecells contained within the bone pastes, when present. For example,encapsulated hUCMSCs differentiate down the osteogenic lineage, asdemonstrated by elevated alkaline phosphatase (ALP) and osteocalcin (OC)gene expression, ALP protein synthesis, and mineralization. Expressionof osteogenic markers and mineralization of hUCMSCs in the injectableCPC-based bone pastes matches those in hydrogel without CPC.

Provided below are specific examples of the injectable bone pastes ofthe present invention. One of ordinary skill in the art will understandthat variations of the injectable bone pastes exemplified herein arewithin the spirit and scope of the invention. For example, as describedbelow, cell types other than hUCMSCs (e.g., but not limited to, stemcells such as hBMSCs and hESCs) can be used in bone pastes comprisingcell-encapsulating microbeads. In addition, the size of the microbeadscan be varied. For example, the microbeads used in the Examples belowhave a mean length of approximately 335 μm and a mean width ofapproximately 232 μm. By varying the air pressure used, the inventorshave produced microbeads of mean widths and lengths of from 100 μm to1500 μm, which are also suitable for injection and creation ofmacropores in CPC after microbead dissolution. Porogenic factors can beincluded in the CPC to also control the porosity of the bone pastes. Thespecific Examples described herein employ alginate-fibrin to fabricatethe cell-encapsulating microbeads; however, other hydrogels such asphoto-cured hydrogels can be used to make cell-encapsulatingmicrobeads-CPC pastes. While the CPCs described in the Examples use TTCPand DCPA, the cell-encapsulating microbeads can be readily incorporatedinto other calcium phosphate cements with various chemistry andcompositions. The CPCs described in the Examples employ a TTCP:DCPAmolar ratio of 1:1 or 1:3. Other TTCP:DCPA ratios (e.g., but not limitedto 1:2), and other fiber types, lengths, and volume fractions can beused in the injectable bone pastes for various orthopedic and other bonerepair/remodeling applications. Additional examples of variations thatcan be employed in the bone pastes described herein are set forth below.

Calcium Phosphate Cement Compositions

The CPCs used in the bone pastes of the present invention vary based onthe identity and proportions of calcium phosphate components thatcomprise the CPC. Suitable calcium phosphate components include and arenot limited to: tetracalcium phosphate (TTCP) (Ca₄(PO₄)₂O), dicalciumphosphate anhydrous (DCPA) (CaHPO₄), dicalcium phosphate dihydrate(CaHPO₄.2H₂O), tricalcium phosphate (Ca₃[PO₄]₂), α-tricalcium phosphate(α-Ca₃(PO₄)₂), β-tricalcium phosphate (β-Ca₃(PO₄)₂), octacalciumphosphate (Ca₈H₂(PO₄)₆.5H₂O), amorphous calcium phosphate (Ca₃(PO₄)₂),calcium carbonate (CaCO₃), calcium hydroxide (Ca[OH]₂), andhydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), and mixtures thereof. In one aspect ofthe invention, the CPC comprises TTCP and DCPA. CPCs comprising TTCP andDCPA can contain various molar ratios of TTCP to DCPA. For example,TTCP:DCPA molar ratios can include ratios from about 1:5 to about 5:1,e.g., about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1,about 3:1, about 4:1, about 5:1, about 3:2, about 2:3, about 4:3, about3:4, about 5:4, about 4:5, and others. An example of an acceptable CPCfor use in the bone pastes of the present invention comprises a mixtureof TTCP:DCPA in a 1:1 molar ratio or in a 1:3 molar ratio.

The size of the TTCP and DCPA particles used to produce the bone pastesof the present invention may vary depending on the other components ofthe pastes, and the use to which the paste will be put. However, TTCPand DCPA particles can independently range in size from about 0.1 μm toabout 1 mm, from about 0.5 μm to about 500 μm, from about 1 μm to about100 μm, from about 0.4 μm to about 3 μm, from about 0.5 μm to about 50μm, from about 5 μm to about 500 μm, or from about 0.5 μm to about 250μm, for example. TTCP and DCPA particles can also be used thatindependently have a median size of about 0.05 μm, about 0.1 μm, about0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 17 μm, about 25 μm,about 50 μm, or about 100 μm, for example.

The liquid portion of the CPC can include, for example, water, chitosan,sodium phosphate, hydroxypropyl methylcellulose, and mixtures thereof.

The CPCs used in the bone pastes of the present invention may alsocomprise a porogen. The inclusion of a porogen permits the formation ofmacropores in the CPC. A suitable combination that may serve as aporogen in the CPCs of the present invention is sodium hydrogencarbonate (NaHCO₃) and citric acid (e.g., citric acid monohydrate,C₆H₈O₇. H₂O). The acid-base reaction of citric acid with NaHCO₃ producesCO₂ bubbles in CPC, resulting in a macroporous scaffold. NaHCO₃ may beadded to the CPC powder at a NaHCO₃/(NaHCO₃+CPC powder) mass fractionsof from about 0.5% to about 35%, including from about 5% to about 30%,from about 7.5% to about 25%, and from about 10% to about 20%. NaHCO₃may also be added to the CPC powder at a NaHCO₃/(NaHCO₃+CPC powder) massfraction of about: 5%, 7.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 22.5%, 25%, 27.5%, and 30%. An amount of citric acidsufficient to maintain a fixed NaHCO₃/(NaHCO₃+C₆H₈O₇. H₂O) mass fractionof from about 20% to about 70%, including from about 40% to about 60%,and from about 50% to about 60%. Particular examples include a massfraction of about: 52.5%, 53%, 53.5%, 54%, 54.5%, 54.52%, 55%, 55.5%,56%, and 56.5%. Additional porogenic factors that may be used includesugar particles, mannitol particles, salt particles and other agentsthat can dissolve and degrade to create voids in CPC to increase theporosity.

When forming the bone pastes of the present invention, various ratios ofCPC powder to liquid mass can be used, including, but not limited to,1:5 to about 5:1, e.g., about 1:5, about 1:4, about 1:3, about 1:2,about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 3:2, about2:3, about 4:3, about 3:4, about 5:4, about 4:5, and others.

Biofunctional Agents

In addition to CPC, the bone pastes of the present invention include atleast one biofunctional agent selected from the following five classesof biofunctional agents.

The first class is the RGD (Arg-Gly-Asp)-containing peptides. Thesepeptides contain a known integrin-recognition site (RGD) to promote cellattachment [36,36]. RGD is the principle integrin-binding domain presentin ECM proteins and it is able to bind multiple integrin species. Inaddition to RGD peptide itself, RGD-containing peptides include, but arenot limited to, the oligopeptides having the following sequences:(Glycine)4-Arginine-Glycine-Aspartic Acid-Serine-Proline (G4RGDSP),Arg-Gly-Asp-Ser (RGDS), Gly-Arg-Gly-Asp (GRGD), Gly-Arg-Gly-Asp-Gly-Tyr(GRGDGY), Arg-Gly-Asp-Ser-Gly-Gly-Cys (RGDSGGC),and Gly-Arg-Gly-Asp-Ser(GRGDS).

The second class is fibronectin (Fn), which is a general cell adhesionmolecule that anchors cells to collagen and proteoglycan [40,41]. Fn isalso an important ECM protein. It regulates many cellular functions anddirects cell adhesion, proliferation, and differentiation via directinteractions with cell surface integrin receptors [40,41]. Fn issynthesized by adherent cells which assemble into a fibrillar networkthrough integrin-dependent and fibronectin-integrin interactions [42].

The third class is fibronectin-like engineered protein polymer (FEPP),which has also been shown to enhance cell adhesion [45-47]. FEPP isgenetically-engineered to include 13 copies of the cell attachmentepitope of human fibronectin between repeated structural peptide units.It has a stable three-dimensional conformation resistant to thermal andchemical denaturation.

The fourth class is extracellular matrix (ECM). Extracellular matrices(ECMs) can enhance embryonic stem cell culture [48-51]. 3D basementmembrane ECMs, such as GELTREX™ (Invitrogen), contain a number ofbioactive molecules that enhance cell functions [51]. GELTREX™ is asoluble form of reduced growth factor basement extract, consistingmainly of laminin, collagen IV, entactin, and heparin sulfateproteoglycan, according to the manufacturer. The ECMs that may be usedas biofunctional agents also include, but are not limited to, humanextracellular matrix (derived from human placenta; BD Biosciences),Matrigel (BD Biosciences), HydroMatrix Peptide Hydrogel (Sigma), andPuraMatrix Peptide Hydrogel (BD Biosciences).

The fifth class is platelet concentrate, which is a fraction of theplasma in which platelets are concentrated [52-54]. It can be obtainedby withdrawing blood from the vein of the patient. Platelet concentratecontains a mixture of growth factors which play an important role inwound healing and tissue regeneration [43-45]. Recently, an increasingtrend has emerged in the use of autologous platelets to facilitatehealing [57]. Platelets have many bioactive proteins responsible forattracting macrophages, MSCs and osteoblasts, which promote removal ofnecrotic tissue and enhance tissue regeneration and healing. From aninitial 30-60 mL of blood withdrawn from a vein, 3-6 mL of platelet-richplasma can be collected, hence self-production of platelet-rich plasmacan be achieved [59,60]. The platelet concentrates that may be used asbiofunctional agents include, but are not limited to, autologousplatelet concentrate and platelet concentrate from a donor. When theplatelet concentrate is from a donor, tissue typing may be performed toensure compatibility.

The bone pastes of the present invention may include one, two, three,four or all five classes of the biofunctional agents. Thus, bone pastesof the present invention include those having one class of thebiofunctional agent, such as CPC-RGD, CPC-Fn, CPC-FEPP, CPC-ECM, andCPC-Platelets; those having two classes of the biofunctional agent, suchas CPC-RGD-Fn, CPC-RGD-FEPP, CPC-RGD-ECM, CPC-RGD-Platelets,CPC-Fn-FEPP, CPC-Fn-ECM, CPC-Fn-Platelets, CPC-FEPP-ECM,CPC-FEPP-Platelets, and CPC-ECM-Platelets; those having three classes ofthe biofunctional agent, such as CPC-RGD-FEPP-Fn, CPC-RGD-ECM-Fn,CPC-RGD- Platelets-Fn, CPC-RGD-ECM-FEPP, CPC-RGD-Platelets-FEPP,CPC-RGD-Platelets-ECM; those having four classes of the biofunctionalagent, such as CPC-RGD-Fn-FEPP-ECM, CPC-RGD-FEPP-ECM-Platelets,CPC-RGD-ECM-Platelets-Fn, and CPC-RGD-Platelets-Fn-FEPP; those havingall five classes of the biofunctional agent, such asCPC-RGD-Fn-FEPP-ECM-Platelets.

Bone pastes comprising one or more biofunctional agents can be producedby first mixing the agents in the liquid fraction, such as water, andthen mixing in CPC powder. Furthermore, RGD peptide can be covalentlylinked to chitosan, and then dissolved in water, following by mixing inof the CPC powder, when chitosan is included in the bone paste.

The amount of a particular biofunctional agent that is include in a bonepaste will vary based on such factors as the identity of the compoundsforming the bone paste; the presence or absence of chitosan, fibers, andporogens; the presence or absence of cells or cell-encapsulatingmicrobeads; the identity of the cells; the identity of the microbeads,and the intended use of the bone paste. However, the amount ofRGD-containing peptide in the bone paste, when present, will generallybe between about 0.0005% and about 5% by mass, or between about 0.001%and about 1% by mass, but is not limited to these ranges. The amount ofRGD-containing peptide in the bone paste, when present, can also beconsidered to be about 0.0005% by mass, about 0.001% by mass, about0.005% by mass, about 0.01% by mass, about 0.05% by mass, about 0.1% bymass, about 0.5% by mass, or about 1% by mass, but is not limited tothese values.

The amount of fibronectin in the bone paste, when present, willgenerally be between about 0.0005% and about 5% by mass, or betweenabout 0.001% and about 1% by mass, but is not limited to these ranges.The amount of fibronectin in the bone paste, when present, can also beconsidered to be about 0.0005% by mass, about 0.001% by mass, about0.005% by mass, about 0.01% by mass, about 0.05% by mass, about 0.1% bymass, about 0.5% by mass, or about 1% by mass, but is not limited tothese values.

The amount of FEPP in the bone paste, when present, will generally bebetween about 0.0005% and about 5% by mass, or between about 0.001% andabout 1% by mass, but is not limited to these ranges. The amount of FEPPin the bone paste, when present, can also be considered to be about0.0005% by mass, about 0.001% by mass, about 0.005% by mass, about 0.01%by mass, about 0.05% by mass, about 0.1% by mass, about 0.5% by mass, orabout 1% by mass, but is not limited to these values.

The amount of ECM in the bone paste, when present, will generally bebetween about 0.01% and about 10% by mass, or between about 0.05% andabout 5% by mass, but is not limited to these ranges. The amount of ECMin the bone paste, when present, can also be considered to be about0.01% by mass, about 0.05% by mass, about 0.1% by mass, about 0.5% bymass, about 1% by mass, about 2% by mass, about 3% by mass, about 4% bymass, or about 5% by mass, but is not limited to these values.

The amount of platelet concentrate in the bone paste, when present, willgenerally be between about 0.01% and about 10% by mass, or between about0.05% and about 5% by mass, but is not limited to these ranges. Theamount of platelet concentrate in the bone paste, when present, can alsobe considered to be about 0.01% by mass, about 0.05% by mass, about 0.1%by mass, about 0.5% by mass, about 1% by mass, about 2% by mass, about3% by mass, about 4% by mass, or about 5% by mass, but is not limited tothese values.

When forming the bone pastes of the present invention, the biofunctionalagent(s) is mixed with the liquid fraction first, which may includechitosan and/or fibers and/or porogens, and then the CPC powder is mixedin.

Chitosan

The CPCs of the present invention may also contain chitosan. Chitosancontent can vary from about 0% to about 50% by mass, for example (butnot limited to), 0% to about 5%; about 5 to about 10%; about 10% toabout 15%; about 15 to about 20%; about 20 to about 25%; about 25 toabout 30%; about 30 to about 35%; about 35 to about 40%; about 40 toabout 45%; about 45 to about 50%, by mass, e.g., 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,30%, 35%, 40%, 45%, 50%. Suitable chitosan includes chitosan lactate(Vanson, Redmond, Wash.). One means for producing the bone pastes of thepresent invention is to dissolve chitosan, such as chitosan lactate, inwater to form “chitosan liquid.” For example, chitosan lactate can bedissolved in water at a chitosan/(chitosan+water) mass fraction of about15%. Into the chitosan liquid can then be mixed the fibers and porogens,when present, and the biofunctional agents, followed by CPC powder.

When RGD-containing peptides are included as a biofunctional agent inthe bone pastes of the present invention, they may be simply mixed intothe chitosan liquid as described above. Alternatively, theRGD-containing peptides may be covalently conjugated to chitosan (RGDgrafting), and then dissolved in water to obtain the chitosan liquid.EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) in combination withsulfo-NHS (N-hydroxysuccinimide) may be used as carboxyl activatingagents for the coupling of primary amines to yield amide bonds.

Fibers

The CPCs of the present invention may also contain fibers to strengthenand/or reinforce the bone pastes. Fibers that can be used in the bonepastes of the invention include and are not limited to: rods, fibers,ropes, threads, or meshes. The fibers can be, e.g., glass fibers,ceramic fibers, polymer fibers, metal fibers, or mixtures thereof.Examples include and are not limited to: Type I bovine collagen fibers,poly(L-lactide)-based polymer fibers, glycolic acid-based polymers, andpoly(D,L-lactic acid) fibers. The fiber diameters can range from about100 nm to about 1 mm. The length can range from about 0.1 mm to about 10mm. The fibers can be non-degradable or degradable. The amount of fibersin the CPC may be based on the volume fraction (vol %) of fibers andincludes, e.g., about 0.5% to about 50%, e.g., about: 0.5-5, 5-10,10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, and 45-50 vol %, andabout: 5 vol %, 10 vol %, 12.5 vol %, 15 vol %, 17.5 vol %, 20 vol %,22.5 vol %, 25 vol %, 27.5 vol %, 30 vol %, 32.5 vol %, 35 vol %, 37.5vol %, 40 vol %, 42.5 vol %, 45%, 47.5 vol %, and 50 vol %. The amountof fibers in the CPC may also be based on the mass fraction % of fibersand includes, e.g., about 0.5% to about 50%, e.g., about: 0.5-5, 5-10,10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, and 45-50%, and about:5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%,37.5%, 40%, 42.5%, 45%, 47.5%, and 50%. The concentration of fibers candiffer based on the diameter of the fibers as large-diameter fibers thatare relatively stiff are less injectable, finer fibers are more flexibleand may be more injectable. Vol % equals the volume of fibers/the volumeof the complete bone paste. An example of a suitable fiber is anabsorbable suture fiber, such as Vicryl™, from Ethicon, Somerville,N.J., and Type I bovine collagen fibers.

Bioactive Agents

The CPCs of the present invention may also contain one or more bioactiveagents. There is no particular limitation on the identity of agents thatmay be utilized, but examples include agents that induce migration ofcells to the locus of bone paste application, agents that inducematuration and/or differentiation of cells in the locus of bone pasteapplication, and agents that promote the growth, differentiation,attachment and/or proliferation of the cells encapsulated within thebone paste. Suitable agents include, but are not limited to, cytokines,growth factors, bone morphogenic proteins (e.g. BMP-1, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-7, and BMP-8a), hormones, steroids,anesthetics, analgesics, opioids, anti-inflammatory agents, includinganti-inflammatory steroid or non-steroidal anti-inflammatory agents,enzyme inhibitors, immunosuppressive agents, growth hormone antagonists,radio- and chemo-therapeutic agents, antimicrobial agents, antibiotics,anti-parasite and/or anti-protozoal compounds, muscle relaxants,anti-spasmodics and muscle contractants including channel blockers,miotics and anti-cholinergics, actin inhibitors, remodeling inhibitors,cell growth inhibitors, anti-adhesion molecules, vasodilating agents,anti-pyretics, anti-angiogenic factors, anti-secretory factors,anti-coagulants and/or anti-thrombotic agents, inhibitors of DNA, RNA orprotein synthesis, peptides, proteins, enzymes, lubricants, and imagingagents. In a certain embodiments, the bioactive agent is a drug.

Polymers for Cell Encapsulation

The bone pastes of the present invention may optionally containmicrobeads that encapsulate cells, such as stem cells. Hydrogels andbiocompatible polymers for cell encapsulation include and are notlimited to: alginate, partially oxidized alginate, oxidized alginate,alginate-fibrin, partially oxidized alginate-fibrin, oxidizedalginate-fibrin, poly(ethylene glycol diacrylate), poly(ethyleneglycol)-anhydride dimethacrylate, gelatin, chemically cross-linkedpolymers, ionically cross-linked polymers, heat-polymerized polymers,and photopolymerized polymers.

In one aspect, oxidized alginate-fibrin microbeads are used, whereinfibrinogen is added to an alginate solution at a fibrinogen massfraction of from about 0.05% to about 1% to render microbeads that havesufficient mechanical integrity and that are readably degradable. Thefibrinogen may also be added to the alginate solution at a fibrinogenmass fraction of about: 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%,0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.18%, 0.20%, 0.22%, 0.24%, 0.26%,0.28%, 0.30%, 0.32%, 0.34%, 0.36%, 0.38%, 0.40%, 0.42%, 0.44%, 0.46%,0.48%, 0.50%, 0.52%, 0.54%, 0.56%, 0.58%, 0.60%, 0.62%, 0.64%, 0.66%,0.68%, 0.70%, 0.72%, 0.74%, 0.76%, 0.78%, 0.80%, 0.82%, 0.84%, 0.86%,0.88%, 0.90%, 0.92%, 0.94%, 0.96%, 0.98%, or 1%. Alginate oxidized toabout 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%,7.5%, 8%, 8.5%, 9%, 9.5%, 10%, or more, or less, may be used.

The alginate-fibrin microbeads may be produced by first preparing anoxidized alginate solution by dissolving sodium alginate in water, andadding sodium periodate to induce an oxidization reaction.Ethanol-precipitated product is dissolved in saline, and fibrinogen isadded to the solution to yield a mixed alginate-fibrinogen solution.Cells are then added to the oxidized alginate-fibrinogen solution, suchas at a density of 1×10⁶ cells/mL. The alginate-fibrinogen droplets areproduced as described in PCT/US11/34457 and sprayed into a solution of acalcium chloride and thrombin, where the calcium chloride inducesalginate cross-linking, while a reaction between fibrinogen and thrombinproduces fibrin.

Hydrogels and biocompatible polymers for cell encapsulation can beformed into microbeads of various diameters for use in the bone pastesdescribed herein. For example, average microbead diameters including butnot limited to about 50 μm to about 1500 μm can be used, e.g., about: 50μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, andvarious ranges and mixtures thereof. Smaller and larger bead sizes maybe used, as long as they the beads can be easily injected withoutcompromising cell viability. In general, beads should be less than 2millimeters, so as to minimize cell damage caused by injection of thebone paste.

Examples of volume fractions (vol %) of microbeads for use in the bonepastes described herein are (and are not limited to): about 10% to about80%, e.g., about: 40-45%, 45-50%, 50-52%; 50-55%, 55-60%, 40-50%,50-60%, 55-65%, 60-70%, 65-75%, 70-80%, and about: 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, and the like. Vol % equals the volume ofmicrobeads/the volume of the complete bone paste.

The length of time required for microbead degradation and release of theencapsulated cells can be varied depending on the material used toprepare the beads. In some applications, maintaining the cells withinthe microbeads for a longer period, such as multiple days, weeks, oreven months, can be desirable, while in other applications quick release(within hours or a small number of days) can be preferable. Whenshort-term release of cells is required, alginate-fibrin microbeads maybe used. Alginate microbeads comprising a small amount of added fibrinhave dramatically increased degradation and decreased therelease-of-cell times in culture and inside the bone paste. Further,when the cells were released from the degraded microbeads and attachedto CPC, they showed a healthy spreading and spindle morphology,consistent with previous studies on cell attachment on CPC surfaces[33].

Cells

Cells that can be encapsulated in the bone pastes and microbeads of theinvention include and are not limited to: bone-growing cells, bloodvessel-growing cells, and cartilage-growing cells, such as mesenchymalstem cells, embryonic stem cells, umbilical cord stem cells, bone marrowstem cells, lymphoid stem cells, myeloid stem cells, stromal cells,osteogenic cells, osteoblast cells, chondrogenic cells, angiogeniccells, endothelial cells, and mixtures thereof. The noted stem cellsinclude totipotent stem cells, pluripotent stem cells, inducedpluripotent stem cells, multipotent stem cells, oligopotent stem cells,unipotent cells, progenitor cells, and osteoblasts. Cells can be humanor from any other suitable animal. Breast milk can be used as a sourceof the stem cells.

Various cell densities can be used in the bone pastes and in theconstruction of the microbeads encapsulating the cells, e.g. but notlimited to, about 10⁴ cells/ml of polymer solution to about 5×10⁶cells/ml of polymer solution, e.g., about: 10⁴, 10⁵, 5×10⁵, 10⁶,2.5×10⁶, 5×10⁶, etc. E.g., one useful range of cell densities is about5×10⁵ to about 2×10⁶, e.g., about 10⁶. After injection of a bone pastecontaining the cells, cell viability is preferably at least about 50%,e.g, at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and thelike, compared to cell viability before injection.

Bone Paste Kits

The bone pastes of the present invention may be used in a variety ofapplications and settings. It is clear that one setting is a physician'soffice or an operating room where the paste is applied to a site of boneinjury. The bone pastes may be provided in kits for use in suchcircumstances where components of the bone pastes are included in thekit and combined on site. The components of such kits may include:

1) A powder and a liquid, which include CPC, one or more biofunctionalagents, and optionally, one or more of chitosan, fibers and a porogen

2) Cell-encapsulating microbeads (either from a frozen stock, or freshlyencapsulated)

3) Mixing pad, spatula, and an injection syringe system

It will be clear that kits comprising variations on the listedcomponents are encompassed within the scope of the invention and thatsuch variations will be readily apparent to the skilled artisan.

Specific embodiments of the present invention are more particularlydescribed in the following Examples, which are intended as illustrativeonly, since numerous modifications and variations thereof will beappreciated by those of ordinary skill in the art.

EXAMPLES Example 1 1A. Materials and Methods

1.1 Fabrication of biofunctionalized CPC Tetracalcium phosphate [TTCP:Ca₄(PO₄)₂O] was synthesized using equimolar amounts of dicalciumphosphate anhydrous (DCPA: CaHPO₄) and calcium carbonate (J. T. Baker,Philipsburg, N.J.). TTCP was ground to obtain particles of 1 to 80 μm,with a median of 17 μm. DCPA was ground to obtain a median particle sizeof 1 μm. TTCP and DCPA powders were mixed at 1:1 molar ratio to form theCPC powder. Chitosan lactate (Vanson, Redmond, Wash.) was mixed withwater at a chitosan/(chitosan+water) mass fraction of 15% to form theliquid, which could cause fast-setting to the CPC paste [55]. Formechanical reinforcement, a resorbable suture fiber (Vicryl, polyglactin910, Ethicon, NJ) was cut to filaments of a length of 3 mm and mixedwith CPC paste at a fiber volume fraction 20%, following a previousstudy [55]. The CPC powder to liquid mass ratio of 2:1 was used to forma flowable paste. This CPC is referred to as “CPC control”.

Five biofunctionalized CPCs were made by incorporating the followingbiofunctional agents: RGD, Fn, FEPP, GELTREX™, and platelet concentrate.Each biofunctional agent was mixed with the chitosan liquid, which wasthen mixed with the CPC powder. The concentration of RGD (Sigma, St.Louis, Mo.) was 50 μg RGD per 1 g of CPC paste (0.005% by mass),following a previous study [38]. For Fn (human plasma Fn, Invitrogen,Carlsbad, Calif.) and FEPP (Sigma), the same 0.005% concentration wasused in CPC. GELTREX™ (Invitrogen) was added to CPC at 100 μL GELTREX™per 1 g of CPC paste (0.1% by mass). This concentration was chosenbecause preliminary study showed that it did not adversely affect CPCsetting time and mechanical property, while greatly improving cellfunction. Similarly, human platelet concentrate (1.2×10⁶ platelets perμL, Biological Specialty Corp., Colmar, Pa.) was added to CPC at 100 μLof platelet concentrate per 1 g of CPC paste (0.1% by mass). CPCcontaining these agents are referred to as CPC-RGD, CPC-Fn, CPC-FEPP,CPC- GELTREX™, and CPC-Platelets, respectively.

1.2 Setting Time and Mechanical Properties of Biofunctionalized CPC

Setting time of CPC was measured using a method as previously described[56]. Briefly, CPC paste was filled into a mold of 3×4×25 mm and placedin a humidor at 37° C. At one minute intervals, the specimen wasscrubbed gently with figures until the powder component did not comeoff, indicating that the setting reaction had occurred sufficiently tohold the specimen together. The time measured from the powder-liquidmixing to this point was used as the setting time for the specimen [55].Three specimens were measured for each material (n=3).

To measure mechanical properties, the paste was placed into a mold of3×4×25 mm. The specimens were incubated at 37° C. for 4 h in a humidor,and then demolded and immersed in water at 37° C. for 20 h. Thespecimens were then fractured in three-point flexure with a span of 20mm at a crosshead speed of 1 mm/min on a Universal Testing Machine(5500R, MTS, Cary, N.C.). Flexural strength and elastic modulus weremeasured (n=6) [35,56].

1.3 hUCMSC Culture

hUCMSCs (ScienCell, Carlsbad, Calif.) were derived from the Wharton'sJelly in umbilical cords of healthy babies and harvested as previouslydescribed [11,16]. The use of hUCMSCs was approved by the University ofMaryland. Cells were cultured in a low-glucose Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and1% penicillin-streptomycin (Invitrogen), which is referred to as thecontrol media. Passage 4 cells were used. The osteogenic media had 100nM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and10 nM 1α,25-Dihydroxyvitamin (Sigma) [16,34,35]. A previous studyperformed immunophenotyping of the hUCMSCs using a flow cytometry method[35]. The hUCMSCs expressed a number of cell surface markers (CD29, CD44, CD 105, HLA-class I) characteristic of MSCs, and were negative forendothelial marker (CD31) and typical hematopoietic markers (CD34, CD45). The cells were also negative for HLA Class II [35].

1.4 hUCMSC Adhesion and Proliferation

The materials for the preparation of CPC specimens were sterilized in anethylene oxide sterilizer (Andersen, Haw River, N.C.) for 12 h and thendegassed for 7 d. Each CPC paste was filled into a disk mold with adiameter of 12 mm and a thickness of 1.5 mm. The specimens wereincubated at 37° C. for 1 d. Each CPC disk was placed in a well of a24-well plate, and 50,000 cells in osteogenic medium was added to eachwell. After 1, 4, and 8 d, the constructs were washed in Tyrode's Hepesbuffer, live/dean stained and viewed by epifluorescence microscopy(TE2000S, Nikon, Melville, N.Y.) [35]. Three randomly-chosen fields ofview were photographed for each disk. Five disks yielded 15 photos foreach material at each time point. N_(Live) is the number of live cells,and N_(Dead) is the number of dead cells. Live cell density, D, is thenumber of live cells attached to the specimen divided by the surfacearea A: D=N_(Live)/A [34,35]. The percentage of live cells isP=N_(Live)/(N_(Live)+N_(Dead)).

1.5 Immunofluorescence of Actin Fibers in hUCMSCs on BiofunctionalizedCPC

Actin fibers in the cell cytoskeleton were examined to determine if theaddition of biofunctional agents in CPC would enhance cell attachmentand increase the amount of actin stress fibers. hUCMSC constructs after1 d culture were washed with PBS, fixed with 4% paraformaldehyde for 20min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with0.1% bovine serum albumin (BSA) for 30 min [35,56]. An actincytoskeleton and focal adhesion staining kit (Chemicon, Temecula,Calif.) was used, which stained actin fibers into a red color. Afterincubating the construct with diluted (1:400) TRITC-conjugatedphalloidin, cell nuclei were labeled with 4′-6-diamidino-2-phenylindole(DAPI) to reveal the nuclei in blue color. Fluorescence microscopy(Nikon) was used to examine the specimens. The fluorescence of actinfibers in hUCMSCs was measured via a NIS-Elements BR software (Nikon).The actin fluorescence was increased when the actins stress fiberdensity was increased.

1.6 Osteogenic Differentiation of hUCMSCs on Biofunctionalized CPC

Quantitative real-time reverse transcription polymerase chain reactionmeasurement (qRT-PCR, 7900HT, Applied Biosystems, Foster City, Calif.)was performed. Cell seeding density of 150,000 cells in osteogenicmedium per well was used. Each disk was placed in a well of a 24-wellplate. The constructs were cultured in osteogenic media for 1, 4, and 8d [35]. The total cellular RNA on the scaffolds was extracted withTRIzol reagent (Invitrogen). RNA (50 ng/μl) was reverse-transcribed intocDNA. TaqMan gene expression kits were used to measure the transcriptlevels of the proposed genes on human alkaline phosphatase (ALP,Hs00758162_m1), osteocalcin (OC, Hs00609452_g1), collagen type I (CollI, Hs00164004), Runx2 (Hs00231692_m1) and glyceraldehyde 3-phosphatedehydrogenase (GAPDH, Hs99999905). Relative expression for each targetgene was evaluated using the 2^(−ΔΔCt) method [57]. The C_(t) values oftarget genes were normalized by the C_(t) of the TaqMan humanhousekeeping gene GAPDH to obtain the ΔC_(t) values. These values weresubtracted by the C_(t) value of the hUCMSCs cultured on tissue culturepolystyrene in the control media for 1 d (the calibrator) to obtain theΔΔC_(t) values [35].

1.7 hUCMSC Mineralization on Biofunctionalized CPC

Alizarin Red S (ARS) staining was used to visualize bone mineralizationby the hUCMSCs [35]. hUCMSCs were seeded on CPC disks and cultured inosteogenic media. After 4 d, 14 d and 21 d, the constructs were stainedwith ARS. After staining, the cells-scaffold constructs were washed withdeionized water for several times with gentle rocking for 5 min for eachwash until no dye extraction in the used water was observed. Anosteogenesis assay (Millipore, Billerica, Mass.) was used to extract thestained minerals and measure the ARS concentration at OD₄₀₅, followingthe manufacturer's instructions. ARS standard curve was done with knownconcentration of the dye. Control scaffolds with the same compositionsand treatment, but without hUCMSC seeding, were also measured. Thecontrol's ARS concentration was subtracted from the ARS concentration ofthe scaffold with hUCMSCs, to yield the net mineral concentrationsynthesized by the cells. The time points of 14 d and 21 d were selectedbecause previous studies found a large increase in calcium content from12 d to 21 d [58].

One-way and two-way ANOVA were performed to detect significant (α=0.05)effects of the variables. Tukey's multiple comparison procedures wereused to group and rank the measured values, and Dunn's multiplecomparison tests were used on data with non-normal distribution orunequal variance, both at a family confidence coefficient of 0.95.

1B. Results

FIG. 1 plots the physical properties of biofunctionalized CPC: (A)cement setting time, (B) flexural strength, and (C) elastic modulus(mean±sd; n=6). The setting time was not significantly increased withthe addition of RGD, Fn, and FEPP, while that with GELTREX™ andPlatelets was slightly increased (p<0.05). The flexural strength wasslightly decreased with the addition of biofunctional agents (p<0.05).The elastic moduli of biofunctionalized CPCs were significantly lowerthan that of CPC control (p<0.05).

FIG. 2 shows representative live/dead staining photos. Live cells werestained green and were numerous. Dead cells were stained red and werefew (not shown). Adding biofunctional agents into CPC increased thenumber of live cells attaching to the specimens at 1 d, compared to CPCcontrol. In addition, hUCMSCs on CPC with biofunctional agents had agreater spreading morphology than those on CPC control. Cellproliferation from 1 d to 8 d appeared to be faster on CPC withbiofunctional agents than that on CPC control.

FIG. 3 plots (A) live cell density, and (B) percentage of live cells. In(A), hUCMSCs proliferated well on all CPC scaffolds, with cell densitygreatly increasing from 1 d to 8 d. The live cell density was increasedby nearly 9-fold from 1 d to 8 d on CPC-RGD, CPC-Fn and CPC-Platelets.At 8 d, live cell density on CPC-RGD, CPC-Fn, CPC-GELTREX™ andCPC-Platelets ranged 600-700 cells/mm², which was an order of magnitudehigher than the 65 cells/mm² on CPC control. In (B), the percentages oflive cells were around 90% at 1 d for all CPCs. There was an increase inthe percentage of live cells over time, reaching about 95% at 8 d.

FIG. 4 shows the immunofluorescence of actin fibers. The actin fibers inthe cell cytoskeleton were stained a red color. The cell nuclei werefluoresced blue. Compared to CPC control in (A), the red fluorescencewas greater in CPC with the five biofunctional agents (B-F), indicatingan increased number of actin stress fibers. Extensive networks of actinstress fibers were observed in CPC containing biofunctional agents. In(G), the area of red fluorescence was measured for each image anddivided by the image area to yield the area fraction. Compared to CPCcontrol, the actin fiber fluorescence area fraction was increased by 5-7fold due to the biofunctional agents in CPC.

Osteogenic gene expressions are plotted in FIG. 5 for: (A) ALP, (B)Runx2, (C) OC, and (D) collagen I. In (A), ALP greatly increased at 8 d.Compared to CPC control, the ALP peak was much higher for all fivebiofunctionalized CPCs. CPC-Platelets had the highest ALP (p<0.05). In(B), Runx2 had a similar trend as ALP, with all five biofunctionalizedCPCs having higher values at 8 d than CPC control. CPC-Platelets had thehighest Runx2. In (C) and (D), both OC and collagen I were greatlyincreased at 8 d. The OC and collagen I peaks were much higher forbiofunctionalized CPCs than CPC control (p<0.05).

hUCMSC mineralization is shown in FIG. 6. ARS stained minerals into ared color. In (A), typical staining photos are shown for CPC control,CPC-Fn and CPC-Platelets at 4 d and 21 d, as examples. A thick clump ofmatrix mineralization synthesized by the cells was observed on at 21 d.ARS staining yielded a red color for CPC without cells, because CPCconsisted of hydroxyapatite minerals. However, for CPC with hUCMSCs, thered staining became much thicker and denser over time. There was a layerof new mineral matrix synthesized by the cells that covered the disk,and the mineral staining increased with the addition of biofunctionalagents in CPC. In (B), as measured by the osteogenesis assay, themineral synthesis by the hUCMSCs at 21 d on CPC-Platelets was 3-foldthat on CPC control.

The results demonstrate that the novel biofunctionalized CPCs of thepresent invention greatly improve hUCMSC attachment, proliferation,osteogenic differentiation and mineralization.

Example 2 2A. Materials and Methods

2.1 hUCMSC Culture

Human umbilical cord mesenchymal stem cells (hUCMSCs) were encapsulatedin hydrogel microbeads. The cells were obtained from ScienCell(Carlsbad, Calif.), and their use was approved by the University ofMaryland. These cells were harvested from the Wharton's Jelly inumbilical cords of healthy babies, using a procedure describedpreviously [63]. The cells were cultured in a low-glucose Dulbecco'smodified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1%penicillin-streptomycin (Invitrogen, Carlsbad, Calif.), which isreferred to as the control media. hUCMSCs at 80-90% confluence weredetached and passaged. Passage 4 cells were used in this study. Theosteogenic media consisted of the control media plus 100 nMdexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and 10nM 1α,25-Dihydroxyvitamin (Sigma, St. Louis, Mo.) [64,65].

2.2 Synthesis of Hydrogel Microbeads with hUCMSC Encapsulation

Alginate is non-cytotoxic and can form an ionically-crosslinked networkunder mild conditions without damaging the encapsulated cells [65-67].Alginate was oxidized to increase its degradability. Oxidation was doneusing sodium periodate at the correct stoichiometric ratio of sodiumperiodate/alginate to have certain percentages of alginate oxidation[61]. The percentage of oxidation (%) was the number of oxidized uronateresidues per 100 uronate units in the alginate chain. In a previousstudy, alginate of up to 5% oxidation was synthesized [61]. In ourpreliminary studies, the microbeads using 5% oxidized alginate failed todegrade after 21 d in the culture media. This slow degradation may bedesirable for other applications, but is too slow for releasing cells ina CPC scaffold. The microbeads using 10% oxidized alginate were too weakto be handled. Hence, in the present study, alginate at 7.5% oxidationwas synthesized. The alginate oxidation followed previous procedures[61]. Briefly, 1% by mass of sodium alginate (UP LVG, 64% guluronicacid, MW=75,000-220,000 g/mol, ProNova, Oslo, Norway) was dissolved indistilled water. 1.51 mL of 0.25 mol/L sodium periodate (Sigma) wasadded to 100 mL of alginate solution, which was stirred to react in thedark at room temperature. At 24 h, the oxidization reaction was stoppedby adding 1 g of ethylene glycol and then 2.5 g of sodium chloride.Ethanol of 200 mL was added to precipitate the product, which was thencollected by centrifugation. The precipitates were re-dissolved in 100mL of water and precipitated with 200 mL of ethanol. The secondprecipitates were collected and dissolved in 30 mL of water. The finalproduct was freeze dried for 24 h, and used to make the microbeads.

The oxidized alginate was dissolved in saline at a concentration of 1.2%[62]. Fibrin was added to the oxidized alginate to obtain oxidizedalginate-fibrin microbeads. Fibrinogen from bovine plasma (Sigma) wasadded at a concentration of 0.1% to the alginate solution and incubatedat 37° C. for 2 h to yield a mixed alginate-fibrinogen solution. Thefibrinogen concentration of 0.1% was selected because in preliminarystudies, fibrinogen>0.1% yielded microbeads that were sticking to eachother because fibrin was sticky. Fibrinogen concentration<0.1% resultedin microbeads that were not fast degradable. hUCMSCs were added to thealginate-fibrinogen solution at a density of 1×10⁶ cells/mL. Thealginate-cell solution was loaded into a syringe which was connected toa bead-generating device (Var J1, Nisco, Zurich, Switzerland). Nitrogengas was fed to the gas inlet and a pressure of 8 psi was established toform a coaxial air flow to break up the alginate droplets. To cross-linkthe microbeads, a solution containing 125 mL of 100 mmol/L calciumchloride plus 125 NIH units of thrombin (Sigma) was prepared. When thealginate-fibrinogen droplets were sprayed into this solution, calciumchloride caused the alginate to crosslink, while the reaction betweenfibrinogen and thrombin produced fibrin. This yieldedhUCMSC-encapsulating, oxidized alginate-fibrin microbeads. A recentstudy showed that the alginate-fibrin microbeads thus obtained wereslightly elongated in shape [62]. The measurement of 100randomly-selected microbeads showed a length range of 87-580 μm(mean=335 μm), and the width range of 75-345 μm (mean=232 μm) [62].These oxidized alginate-fibrin microbeads are referred to as“microbeads”.

2.3 hUCMSC-Encapsulating Microbeads in CPC Surface

The hydrogel microbeads were used to protect the encapsulated cells fromthe CPC paste mixing forces and the setting reaction. After CPC setting,the purpose was for the microbeads to degrade and release the cells. Toexamine the effectiveness, the alginate-fibrin microbeads with hUCMSCsencapsulation were seeded into the surface layer of the CPC paste.

The CPC powder consisted of a mixture of tetracalcium phosphate (TTCP),Ca₄(PO₄)₂O, and dicalcium phosphate anhydrous (DCPA), CaHPO₄. TTCP wassynthesized from a reaction between DCPA and CaCO₃, and ground to obtainTTCP particles of 1 to 80 μm, with a median of 17 μm. DCPA was ground toobtain particles of 0.4 to 3.0 μm, with a median of 1.0 μm. TTCP andDCPA were mixed at 1:3 molar ratio to form the CPC powder [67]. Type Ibovine collagen fiber (Sigma) was added to CPC at a mass fraction of5.0% collagen/(collagen+CPC) because a previous study showed thatcollagen in CPC enhanced cell attachment [68]. The CPC liquid consistedof chitosan lactate (Vanson, Redmond, Wash.) dissolved in water at achitosan/(chitosan+water) mass fraction of 15% [69]. Chitosan and itsderivatives are natural biopolymers that are biodegradable andosteoconductive [70], and can impart fast-setting to CPC [55]. A CPCpowder to liquid mass ratio of 2:1 to form the CPC paste. The CPC pastewas filled into a disk mold of 15 mm diameter and 2 mm height, and thepaste surface was flattened with a glass slide. Then, 0.2 mL ofhUCMSC-encapsulating microbeads were placed on the CPC paste and gentlypressed to be partially embedded into the paste. After incubating for 10min at 37° C. and 100% humidity, CPC was initially hardened, and theconstruct was transferred into a 6-well plate. Five mL of osteogenicmedia was added in each well. After 1 d, CPC was fully set. Themicrobead-CPC construct is schematically shown in FIG. 7A.

2.4 Development of Novel Biofunctionalized CPC

Preliminary study indicated that after the hUCMSCs were released fromthe microbeads, the cell attachment on CPC surface was relatively poorand not robust. Therefore, biofunctional molecules were incorporatedinto CPC to improve cell attachment and function. Four differentcompositions were tested: CPC control (no addition of biofunctionalmolecules), CPC mixed with fibronectin, CPC mixed with RGD, and CPCgrafted with RGD.

Fibronectin (Fn) from bovine plasma (Sigma) was mixed with the chitosanliquid, which was then mixed with the CPC powder. The reason for usingFn was that Fn was shown to improve cell attachment to scaffolds[71-73]. Based on our preliminary study, 0.25 mg of Fn was mixed in eachCPC disk, which was shown to greatly enhance cell function. Thisbiofunctionalized CPC is referred to as “CPC-mixed-Fn”.

The tripeptide Arg-Gly-Asp (RGD) is an important functional andcell-binding domain [73-75,80,81]. RGD (Sigma) was mixed with thechitosan liquid, which was then mixed with CPC. As for Fn, 0.25 mg ofRGD was mixed into each CPC disk. This biofunctionalized CPC is referredto as “CPC-mixed-RGD”.

To further improve cell attachment, instead of simply mixing the RGDinto the CPC paste, RGD grafting was performed. Oligopeptides with asequence of (Glycine)4-Arginine-Glycine-Aspartic Acid-Serine-Proline(abbreviated as G4RGDSP) (Peptides International, Louisville, Ky.) werecovalently conjugated to chitosan and then mixed with CPC. EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) in combination withsulfo-NHS (N-hydroxysuccinimide) were used as carboxyl activating agentsfor the coupling of primary amines to yield amide bonds. Because EDC isa zero-length crosslinker, polypeptide like G4RGDSP with RGD as thefunctional sequence is frequently used as cell adhesion peptides forbio-scaffold grafting by carbodiimide chemistry [76,77,80,81]. Briefly,chitosan was dissolved in 0.1 mol/L MES buffer (Sigma) at a massfraction of 1%. EDC (Sigma), Sulfo-NHS (Sigma), and G4RGDSP peptide wereadded to the dissolved chitosan at a molar ratio ofG4RGDSP/EDC/NHS=1/1.2/0.6, and allowed to react for 24 h. The productswere dialyzed against distilled water using a cellulose membrane(MWCO=25 kDa) for 3 d and then freeze dried. This resulted in theRGD-grafted chitosan, which was then dissolved in water at a massfraction of 15% to obtain the chitosan liquid for mixing with CPCpowder. The molecular weight (MW) of G4RGDSP is 759, which is about 2times the MW (346) of RGD. To use an equivalent amount of the RGDsequence, 0.5 mg of G4RGDSP was immobilized in each CPC disk. Thisbiofunctionalized CPC is referred to as “CPC-grafted-RGD”.

2.5 Live/Dead Assay of hUCMSCs from Microbeads and Attachment onBiofunctionalized CPC

The hUCMSC-encapsulating microbeads were embedded into the surface ofthe four different types of CPC pastes. The time periods for examinationwere 1 d, 7 d, 14 d, and 21 d, as schematically shown in FIG. 7A-D.

For the live/dead staining, each sample was incubated at 37° C. for 10min with 2 ml Tyrode's Hepes containing 2 μmol/L calcein-AM and 2 μmol/Lethidium homodimer-1 (Molecular Probes, Eugene, Oreg.). This stainedlive cells into a green color and dead cells into a red color [65]. Anepifluorescence microscopy (Eclipse TE2000, Nikon, Melville, N.Y.) wasused to examine the samples. Three randomly-chosen fields of view werephotographed from each well (six wells yielded 18 photos for each timepoint). The live and dead cells were counted. The percentage of livecells was: P_(Live)=N_(Live)/(N_(Live)+N_(Dead)), where N_(Live)=thenumber of live cells, and N_(Dead)=the number of dead cells. The livecell density, D_(Live), was calculated: D_(live)=N_(Live)/A, where A isthe area of the view field for N_(Live).

2.6 Osteogenic Differentiation of Released Cells on BiofunctionalizedCPC

The above experiments showed that CPC-grafted-RGD had the best cellattachment and proliferation. Hence, CPC-grafted-RGD was selected forosteogenic differentiation experiments. Quantitative real-time reversetranscription polymerase chain reaction (qRT-PCR, 7900HT, AppliedBiosystems, Foster City, Calif.) was used. hUCMSC-encapsulatingmicrobeads seeded in the RGD-grafted CPC surface were cultured for 1, 4,7, 14 and 21 d in osteogenic media. The total cellular RNA of the cellswere extracted with TRIzol reagent (Invitrogen) and reverse-transcribedinto cDNA using a High-Capacity cDNA Archive kit. TaqMan gene expressionassay kits, including two pre-designed specific primers and probes, wereused to measure the transcript levels of the proposed genes on humanalkaline phosphatase (ALP, Hs00758162_m1), osteocalcin (OC,Hs00609452_g1), collagen type I (Coll I, Hs00164004), runt-relatedtranscription factor 2 (Runx2, Hs00231692_m1), and glyceraldehyde3-phosphate dehydrogenase (GAPDH, Hs99999905). Relative expression foreach gene was evaluated using the 2-ΔΔC_(t) method [34]. C_(t) values oftarget genes were normalized by the C_(t) of the TaqMan humanhousekeeping gene GAPDH to obtain the ΔC_(t) values. The C_(t) ofhUCMSCs cultured on tissue culture polystyrene in the control media for1 d served as the calibrator [34,65].

2.7 Mineral Synthesis via hUCMSCs

Mineral synthesis via hUCMSCs released from the microbeads andproliferated on CPC-grafted-RGD was measured. At 4, 7, 14 and 21 d(n=5), Alizarin Red S (ARS) staining was performed to visualize bonemineralization [78]. The CPC-grafted-RGD samples seeded withhUCMSC-microbeads were washed with PBS, fixed with 10% formaldehyde, andstained with ARS (Millipore, Billerica, Mass.) for 5 min, which stainedcalcium-rich deposits made by the cells into a red color. Controls wereincubated in the same manner using CPC-grafted-RGD but without thehUCMSC-encapsulating microbeads. An osteogenesis assay kit (Millipore)was used to extract the stained minerals and measure the Alizarin Redconcentration at OD405, following the manufacturer's instructions. Thevalue from the control sample without hUCMSCs was subtracted from thesample seeded with hUCMSCs to obtain the net mineral synthesis by thecells. Time periods of up to 21 d were selected because in previousstudies, a great increase in calcium content during in vitro cellcultures was found between 12 d to 21 d [79].

2.8 Mechanical Testing

The purpose of mechanical testing was to investigate the effect ofbiofunctionalization on CPC mechanical properties. Each CPC compositepaste was placed in a rectangular mold of 3×4×25 mm. The specimens wereincubated at 37° C. in a humidor for 4 h, and then demolded and immersedin water for 20 h. A three-point flexural test was used to fracture thespecimens on a Universal Testing Machine (MTS, Eden Prairie, Minn.)using a span of 20 mm at a crosshead speed of 1 mm/min. Flexuralstrength, S=3FL/(2bh²), where F is the maximum load on theload-displacement (F-d) curve, L is span, b is specimen width and h isthickness. Elastic modulus E=(F/d) (L³/[4bh³]). Work-of-fracture(toughness) was calculated as the area under the F-d curve divided bythe specimen's cross-sectional area [68].

One-way and two-way ANOVA were performed to detect significant (α=0.05)effects of the variables. Tukey's multiple comparison tests were used togroup and rank the measured values, and Dunn's multiple comparison testswere used on data with non-normal distribution or unequal variance, bothat a family confidence coefficient of 0.95.

2B. Results

FIG. 7A-D show the schematic of alginate-fibrin microbead partiallyembedded in CPC and cultured for 1 d, 7 d, 14 d and 21 d, respectively,illustrating microbead degradation over time, and cell release onto CPC.Optical photos of the microbeads are shown in (E) and (F), at 1 d and 14d, respectively. Because the microbeads were nearly transparent anddifficult to see, a blue filter was used to enhance the contrast andclarity of the microbeads in (E) and (F). These results indicate thatthe microbeads were degrading over time and the cells were successfullyreleased and migrated out of the macrobeads in (F). Note that the cellsencapsulated inside the microbeads appeared as rounded dots in (E),while in (F) the released cells were spreading and elongated inmorphology.

The live/dead staining photos are shown in FIG. 8 forhUCMSC-encapsulating microbeads embedded in the surface of CPC control,CPC-mixed-Fn, CPC-mixed-RGD, and CPC-grafted-RGD. At 1 d, 7 d, 14 d and21 d, the CPC control had limited amount of live cells, consistent withobservations in our preliminary study. The released cells did not attachwell to CPC, and some cells likely were lost during media change.Noticeable improvements were achieved when Fn or RGD were added to CPC.The cells attached at 7 d and proliferated at 14 d and 21 d. Among thethree bioactive agent treatment, CPC-grafted-RGD had the best cellattachment and the fastest cell proliferation. Dead cells (stained red)were shown in FIG. 8 (last row) for 21 d as an example, and there werefew dead cells in all cases.

FIG. 9 plots (A) the percentage of live cells, and (B) live celldensity, for the hUCMSCs released from the microbeads and attached onCPC containing different bioactive agents. In (A), CPC-mixed-Fn andCPC-mixed-RGD had similar percentages of live cells (p>0.1), and bothwere significantly higher than CPC control (p<0.05). CPC-grafted-RGD hadthe highest percentages of live cells among all the groups (p<0.05). In(B), the number of live cells per CPC surface area (mm²) increased withincreasing culture time due to cell proliferation. Both CPC-mixed-RGDand CPC-mixed-Fn had more live cells than CPC control (p<0.05). Amongthese four groups, CPC-grafted-RGD had dramatically more live cells thanthe rest. At 21 d, compared to CPC control, the live cell density wasincreased by 4-fold via mixing the RGD into CPC, and the increase was9-fold when the RGD was grafted with CPC.

Since CPC-grafted-RGD had the best cell attachment and proliferation,this group was selected for the examination of osteogenicdifferentiation as a function of culture time. The RT-PCR results areplotted in FIG. 10 for (A) ALP, (B) OC, (C) collagen type I, and (D)Runx2 gene expressions. All four markers reached much higher levels than1 d, indicating successful osteogenic differentiation of the hUCMSCsreleased from the microbeads and attached to the CPC-grafted-RGDscaffold.

FIG. 11 shows the mineralization results for hUCMSCs released from themicrobeads and attached to the CPC-grafted-RGD disks. Disks withoutcells were immersed as control for the same time periods, with anexample in (A) at 21 d. CPC contained apatite minerals, hence the diskswithout cells stained a red color. In (B), the disk with cells at 7 dalso stained red. However, when the culture time was increased to 14 dand 21 d (C and D, respectively), an additional red substanceaccumulated on the disks. The red staining became much thicker anddenser over time, and the layer of new mineral matrix synthesized by thecells covered the entire disk at 21 d (D). The thick matrixmineralization by the cells covered not only the top surface, but alsothe peripheral areas at the sides of the scaffold disks at 21 d. Themineral concentration measured by an osteogenesis assay is plotted in(E). The mineral synthesis by the hUCMSCs released from microbeads andattached to CPC-grafted-RGD was minimal at 4 and 7 d, but greatlyincreased at 14 and 21 d (p<0.05).

While adding bioactive agents into CPC improved the function of hUCMSCs,it is important that the CPC mechanical properties are not compromisedin order to use the CPC-stem cell construct in load-bearing repairs.FIG. 12 plots the (A) flexural strength, (B) elastic modulus, and (C)work-of-fracture (toughness) of CPC composites. Mixing Fn or RGD intoCPC did not decrease the mechanical properties of CPC, compared to thatwithout bioactive agents. CPC grafted with RGD had higher mechanicalproperties than the other three materials (p<0.05).

The results show that (1) hUCMSC-encapsulating alginate-fibrinmicrobeads in the surface of biofunctionalized CPC released the cellswhich attached to CPC and differentiated down the osteogenic lineage;and (2) incorporating biofunctional molecules such as RGD and Fn greatlyimproved the cell function on CPC.

INCORPORATIO BY REFERENCE

Throughout this application, various publications, patents, and/orpatent applications are referenced in order to more fully describe thestate of the art to which this invention pertains. The disclosures ofthese publications, patents, and/or patent applications are hereinincorporated by reference in their entireties to the same extent as ifeach independent publication, patent, and/or patent application wasspecifically and individually indicated to be incorporated by reference.

Other Embodiments

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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What is claimed is:
 1. A bone paste comprising calcium phosphate cementand one or more biofunctional agents, wherein the biofunctional agentsare selected from the group consisting of RGD-containing peptides,fibronectin, fibronectin-like engineered polymer protein (FEPP), derivedextracellular matrix (dECM), and platelet concentrate.
 2. The bone pasteof claim 1, wherein the bone paste comprises calcium phosphate cementand an RGD-containing peptide, wherein RGD-containing peptide isselected from the group consisting of RGD, G4RGDSP, RGDS, GRGD, GRGDGY,RGDSGGC, and GRGDS, and wherein RGD-containing peptide is present withina range of about 0.0005% to about 5% by mass.
 3. The bone paste of claim1, wherein the bone paste comprises calcium phosphate cement andfibronectin, wherein fibronectin is present within a range of about0.0005% to about 5% by mass.
 4. The bone paste of claim 1, wherein thebone paste comprises calcium phosphate cement and FEPP, wherein FEPP ispresent within a range of about 0.0005% to about 5% by mass.
 5. The bonepaste of claim 1, wherein the bone paste comprises calcium phosphatecement and dECM, wherein dECM is present within a range of about 0.001%to about 10% by mass.
 6. The bone paste of claim 1, wherein the bonepaste comprises calcium phosphate cement and platelet concentrate,wherein platelet concentrate is present within a range of about 0.001%to about 10% by mass.
 7. The bone paste of claim 1, wherein the bonepaste comprises calcium phosphate cement and any two of thebiofunctional agents.
 8. The bone paste of claim 1, wherein the bonepaste comprises calcium phosphate cement and any three of thebiofunctional agents.
 9. The bone paste of claim 1, wherein the bonepaste comprises calcium phosphate cement and any four of thebiofunctional agents.
 10. The bone paste of claim 1, wherein the bonepaste comprises calcium phosphate cement and each of the fivebiofunctional agents.
 11. The bone paste of any one of claims 1-10,wherein the calcium phosphate cement comprises one or more ingredientsselected from the group consisting of tetracalcium phosphate (TTCP)(Ca₄(PO₄)₂O), dicalcium phosphate anhydrous (DCPA) (CaHPO₄), dicalciumphosphate dihydrate (CaHPO₄.2H₂O), tricalcium phosphate (Ca₃[PO₄]₂),α-tricalcium phosphate (α-Ca₃(PO₄)₂), β-tricalcium phosphate(β-Ca₃(PO₄)₂), octacalcium phosphate (Ca₈H₂(PO₄)₆.5H₂O), amorphouscalcium phosphate (Ca₃(PO₄)₂), calcium carbonate (CaCO₃), calciumhydroxide (Ca[OH]₂), and hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), and mixturesthereof.
 12. The bone paste of any one of claims 1-10, wherein thecalcium phosphate cement comprises tetracalcium phosphate and dicalciumphosphate anhydrous.
 13. The bone paste of claim 12, wherein the calciumphosphate cement comprises a molar ratio of tetracalcium phosphate todicalcium phosphate anhydrous of about 1:5 to about 5:1.
 14. The bonepaste of claim 12, wherein the calcium phosphate cement comprises amolar ratio of tetracalcium phosphate to dicalcium phosphate anhydrousof about 1:3 to about 1:1.
 15. The bone paste of claim 12, wherein thecalcium phosphate cement comprises an approximately 1:1 molar ratio oftetracalcium phosphate to dicalcium phosphate anhydrous.
 16. The bonepaste of any one of claims 1-10, wherein the calcium phosphate cementfurther comprises chitosan.
 17. The bone paste of any one of claims1-10, wherein the calcium phosphate cement further comprises fibers. 18.The bone paste of claim 17, wherein the fibers are degradable.
 19. Thebone paste of any one of claims 1-10, wherein the calcium phosphatecement further comprises chitosan and degradable fibers.
 20. The bonepaste of any one of claims 1-10, wherein the calcium phosphate cementfurther comprises a porogen.
 21. The bone paste of claim 20, wherein theporogen is NaHCO₃ and citric acid.
 22. The bone paste of claim 21,wherein the mass fraction of NaHCO₃ is from about 5% to about 30%, andthe mass fraction of citric acid is from about 50% to about 60%.
 23. Thebone paste of claim 16, wherein the bone paste comprises calciumphosphate cement and a RGD-containing peptide, wherein theRGD-containing peptide is present within a range of about 0.0005% toabout 0.05% by mass, and wherein the RGD-containing peptide iscovalently linked to said chitosan.
 24. The bone paste of any one ofclaims 1-10, wherein the bone paste further comprises stem cells. 25.The bone paste of claim 24, wherein the cells are selected from thegroup consisting of human umbilical cord mesenchymal stem cells, bonemarrow stem cells, embryonic stem cells, pluripotent stem cells, inducedpluripotent stem cells, multipotent stem cells, progenitor cells, andosteoblasts.
 26. The bone paste of claim 24, wherein the cells areattached to a surface of the bone paste, or the cells are interspersedthroughout the bone paste, or both.
 27. The bone paste of any one ofclaims 1-10, wherein the bone paste is injectable.
 28. A bone pastecomprising calcium phosphate cement, one or more biofunctional agentsand cell-encapsulating microbeads, wherein the biofunctional agents areselected from the group consisting of RGD peptide, fibronectin,fibronectin-like engineered polymer protein (FEPP), derivedextracellular matrix (dECM), and platelet concentrate.
 29. The bonepaste of claim 28, wherein the bone paste comprises calcium phosphatecement and an RGD-containing peptide, wherein RGD-containing peptide isselected from the group consisting of RGD, G4RGDSP, RGDS, GRGD, GRGDGY,RGDSGGC, and GRGDS, and wherein RGD-containing peptide is present withina range of about 0.0005% to about 5% by mass.
 30. The bone paste ofclaim 28, wherein the bone paste comprises calcium phosphate cement andfibronectin, wherein fibronectin is present within a range of about0.0005% to about 5% by mass.
 31. The bone paste of claim 28, wherein thebone paste comprises calcium phosphate cement and FEPP, wherein FEPP ispresent within a range of about 0.0005% to about 5% by mass.
 32. Thebone paste of claim 28, wherein the bone paste comprises calciumphosphate cement and dECM, wherein dECM is present within a range ofabout 0.001% to about 10% by mass.
 33. The bone paste of claim 28,wherein the bone paste comprises calcium phosphate cement and plateletconcentrate, wherein platelet concentrate is present within a range ofabout 0.001% to about 10% by mass.
 34. The bone paste of claim 28,wherein the bone paste comprises calcium phosphate cement and any two ofthe biofunctional agents.
 35. The bone paste of claim 28, wherein thebone paste comprises calcium phosphate cement and any three of thebiofunctional agents.
 36. The bone paste of claim 28, wherein the bonepaste comprises calcium phosphate cement and any four of thebiofunctional agents.
 37. The bone paste of claim 28, wherein the bonepaste comprises calcium phosphate cement and each of the fivebiofunctional agents.
 38. The bone paste of any one of claims 28-37,wherein the calcium phosphate cement comprises one or more ingredientsselected from the group consisting of tetracalcium phosphate (TTCP)(Ca₄(PO₄)₂O), dicalcium phosphate anhydrous (DCPA) (CaHPO₄), dicalciumphosphate dihydrate (CaHPO₄.2H₂O), tricalcium phosphate (Ca₃[PO₄]₂),α-tricalcium phosphate (α-Ca₃(PO₄)₂), β-tricalcium phosphate(β-Ca₃(PO₄)₂), octacalcium phosphate (Ca₈H₂(PO₄)₆5H₂O), amorphouscalcium phosphate (Ca₃(PO₄)₂), calcium carbonate (CaCO₃), calciumhydroxide (Ca[OH]₂), and hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), and mixturesthereof.
 39. The bone paste of any one of claims 28-37, wherein thecalcium phosphate cement comprises tetracalcium phosphate and dicalciumphosphate anhydrous.
 40. The bone paste of claim 39, wherein the calciumphosphate cement comprises a molar ratio of tetracalcium phosphate todicalcium phosphate anhydrous of about 1:5 to about 5:1.
 41. The bonepaste of claim 39, wherein the calcium phosphate cement comprises amolar ratio of tetracalcium phosphate to dicalcium phosphate anhydrousof about 1:3 to about 1:1.
 42. The bone paste of claim 39, wherein thecalcium phosphate cement comprises an approximately 1:1 molar ratio oftetracalcium phosphate to dicalcium phosphate anhydrous.
 43. The bonepaste of any one of claims 28-37, wherein the calcium phosphate cementfurther comprises chitosan.
 44. The bone paste of any one of claims28-37, wherein the calcium phosphate cement further comprises fibers.45. The bone paste of claim 40, wherein the fibers are degradable. 46.The bone paste of any one of claims 28-37, wherein the calcium phosphatecement further comprises chitosan and degradable fibers.
 47. The bonepaste of any one of claims 28-37, wherein the calcium phosphate cementfurther comprises a porogen.
 48. The bone paste of claim 47, wherein theporogen is NaHCO₃ and citric acid.
 49. The bone paste of claim 48,wherein the mass fraction of NaHCO₃ is from about 5% to about 30%, andthe mass fraction of citric acid is from about 50% to about 60%.
 50. Thebone paste of claim 43, wherein the bone paste comprises calciumphosphate cement and a RGD-containing peptide, wherein theRGD-containing peptide is present within a range of about 0.0005% toabout 0.05% by mass, and wherein the RGD-containing peptide iscovalently linked to said chitosan.
 51. The bone paste of any one ofclaims 28-37, wherein the microbeads are hydrogel microbeads.
 52. Thebone paste of claim 51, wherein the microbeads are comprised ofalginate, partially oxidized alginate, oxidized alginate,alginate-fibrin, partially oxidized alginate-fibrin, oxidizedalginate-fibrin, poly(ethylene glycol diacrylate), poly(ethyleneglycol)-anhydride dimethacrylate, gelatin, chemically cross-linkedpolymers, ionically cross-linked polymers, heat-polymerized polymers, orphotopolymerized polymers.
 53. The bone paste of claim 51, wherein themicrobeads are alginate-fibrin microbeads.
 54. The bone paste of claim53, wherein the alginate-fibrin microbeads comprise a fibrinogen massfraction of from about 0.05% to about 1%.
 55. The bone paste of claim53, wherein the alginate-fibrin microbeads comprise a fibrinogen massfraction of about 0.1%.
 56. The bone paste of claim 53, wherein thealginate is at about 7.5% oxidation.
 57. The bone paste of any one ofclaims 28-37, wherein the microbeads are present in a volume of about 40to 60%.
 58. The bone paste of any one of claims 28-37, wherein themicrobeads have an average diameter of less than about 2 millimeters.59. The bone paste of any one of claims 28-37, wherein the cells arestem cells.
 60. The bone paste of any one of claims 28-37, wherein thecells are selected from the group consisting of human umbilical cordmesenchymal stem cells, bone marrow stem cells, embryonic stem cells,pluripotent stem cells, induced pluripotent stem cells, multipotent stemcells, progenitor cells, and osteoblasts.
 61. The bone paste of any oneof claims 28-37, wherein the bone paste is injectable.
 62. A method forpreparing a bone paste, comprising covalently linking an RGD-containingpeptide to chitosan to form RGD-grafted chitosan, dissolving theRGD-grafted chitosan in water to form a chitosan liquid, and mixingcalcium phosphate cement into the chitosan liquid, thereby preparing abone paste.
 63. The method of claim 62, wherein the RGD-containingpeptide is selected from the group consisting of RGD, G4RGDSP, RGDS,GRGD, GRGDGY, RGDSGGC, and GRGDS.
 64. The method of claim 62 or 63,wherein the RGD-containing peptide is present within a range of about0.0005% to about 5% by mass.
 65. The method of claim 62 or 63, whereinthe chitosan is chitosan lactate.
 66. The method of claim 62 or 63,wherein a degradable fiber is added to the chitosan liquid prior tomixing calcium phosphate cement into the chitosan liquid.
 67. The methodof claim 62 or 63, wherein the calcium phosphate cement comprises anapproximately 1:1 molar ratio of tetracalcium phosphate to dicalciumphosphate anhydrous.